US20060014930A1 - Synthetic or natural peptides binding protein phosphatase 2A, identification method and uses - Google Patents

Synthetic or natural peptides binding protein phosphatase 2A, identification method and uses Download PDF

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US20060014930A1
US20060014930A1 US10/763,286 US76328604A US2006014930A1 US 20060014930 A1 US20060014930 A1 US 20060014930A1 US 76328604 A US76328604 A US 76328604A US 2006014930 A1 US2006014930 A1 US 2006014930A1
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peptide
seq
sequence
protein
peptides
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Alphonse Garcia
Xavier Cayla
Angelita Rebollo
Gordon Langsley
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Consejo Superior de Investigaciones Cientificas CSIC
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Recherche Agronomique INRA
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Consejo Superior de Investigaciones Cientificas CSIC
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Recherche Agronomique INRA
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Assigned to INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE, CONSEJO SUPERIOR DE INVESTIGACIONES CIENIFICAS, INSTITUT PASTEUR, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE reassignment INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: REBOLLO, ANGELITA, CAYLA, XAVIER, GARCIA, ALPHONSE, LANGSLEY, GORDON
Publication of US20060014930A1 publication Critical patent/US20060014930A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to novel synthetic or natural peptides, in particular for use in treating viral or parasitic infections or in the treatment of tumors, said peptides being less than 30 amino acids in size, preferably less than 20 amino acids, in particular 15 to 20 amino acids, and characterized in that in vitro, they specifically bind a type 2A protein phosphatase holoenzyme or one of its subunits.
  • the invention also relates to a method for identifying said peptides, and to their uses.
  • Cell physiology is partially controlled by modulating protein phosphorylation.
  • the phosphorylation state of cell proteins depends on the antagonist action of protein kinases which phosphorylates them and protein phosphatases which dephosphorylate them.
  • Protein phosphatases are divided into two principal groups: tyrosine phosphatases and serine/threonine phosphatases. Serine/threonine phosphatases are classified into two categories which depend on the specificicity of their substrate and their sensitivity to certain inhibitors, namely type 1 phosphatases (PP1) and type 2 phosphatases (PP2). Type 2 phosphatases are themselves divided into different classes, including phosphatase 2A (PP2A), phosphatase 2B or calcineurine the activity of which is regulated by calcium, and phosphatase 2C (PP2C) the activity of which is regulated by magnesium.
  • P2A phosphatase 2A
  • phosphatase 2B phosphatase 2B or calcineurine the activity of which is regulated by calcium
  • P2C phosphatase 2C
  • type 2A phosphatases are highly conserved during evolution and are potentially involved in regulating many biological processes.
  • PP2A enzymes have been clearly involved in regulating transcription, control of the cell cycle or viral transformation. Further, PP2As are targeted by different viral or parasitic proteins, suggesting a role for PP2As in, host-pathogen interactions.
  • PP2As are oligomeric complexes (holoenzymes) each comprising a catalytic subunit (C) and one or two regulating subunits (A) and (B).
  • the structure of subunit (A) consists of 15 imperfect repeats of a conserved amino acid sequence of 38 to 40 amino acids, certain of which interact with subunits (B) and (C).
  • Subunits (A) and (C), conserved during evolution, constitute the base structure of the enzyme and are expressed constitutively.
  • subunits (B) constitute a family of regulating proteins not connected via a common structure and expressed differentially (Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 1989; 58: 453-508).
  • Protein phosphatase 2As exist in vivo in two classes with different forms: a dimeric form (AC) and a trimeric form (ABC). Subunits (B) regulate phosphatase activity and specificity towards the substrate. The existence of multiple forms of PP2A is correlated with the distinct and varied functions of PP2A in vivo.
  • parainfluenza virus incorporates the protein PKC ⁇ , a protein of cellular origin under the control of PP2A, into its viral particle. This can perturb the phosphoylation of host proteins and facilitate its own replication (De B P, Gupta S, Barnejee A K. Cellular protein kinase C ⁇ regulates human parainfluenza virus type 3 replication. Proc. Natl Acad Sci USA 1995; 92: 5204-8).
  • DNA viruses with transforming power such as papovae or adenoviruses, as well as certain retroviruses such as the type-1 human immunodeficiency virus (HIV-1), code for proteins which interact directly with certain host PP2As. All of those viruses comprise proteins which, although structurally different, interact with certain holoenzymes and modify phosphatase activity.
  • HIV-1 human immunodeficiency virus
  • E4orf4 protein of adenoviruses binds to a heterotrimeric PP2A and more precisely to a regulating subunit (B), which causes a reduction in the transcription of JunB in the infected cell. That effect could play an important role during viral infection by regulating the apoptotic response of infected cells.
  • B regulating subunit
  • the interaction of E4orf4 with PP2A induces apoptosis in transformed cells in a p53-independent manner (Shtrichman R et al, Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells. J Virol 1998; 72: 2975-82).
  • Tumor-generating viruses of the Papovae family including SV40 and polyoma virus, induce cell transformation. It has been shown that PP2A interacts with the “small T” antigen of SV40 or polyoma and with the transforming “middle T” protein of polyoma. Those interactions of viral proteins with PP2A have been clearly involved in viral transformation. Finally, transcriptional regulation, a process normally carried out in the cell by different factors specifically fixing to promoter regulating sequences, probably represents the most important mechanism involved in the control of viral expression by PP2A.
  • PP2A is a negative regulator for numerous transcription factors involved in particular in the processes of cell growth and proliferation, including AP1/SRE, NF- ⁇ B, Sp1 and CREB (Waszinski, B E, Wheat W H, Jaspers S, Peruski L F, J R Lickier R L, Johnson G L, and Klemm D J, Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 1993 13, 2822-34). Viral regulation of those transcription factors would permit modulation of viral transcription.
  • Vpr The viral protein of HIV-1, Vpr, interacts in vitro with PP2A and stimulates the catalytic activity of PP2A (Tung L et al, Direct activation of protein phosphatase 2A0 by HIV-1 encoded protein complex Ncp7: vpr. FEBS Lett 1997; 401: 197-201). Vpr can induce the G2 stoppage of infected cells by inhibiting the activation of the p34cdc2-cycline B complex. Further, Vpr interacts with the transcription factor Sp1 and is a weak trans-activator for transcription of Sp1 dependent HIV-1.
  • the Vpr protein of HIV-1 which is incorporated into the virion, should be involved in vivo in the initiation of viral transcription, a step that is clearly essential for regulating the expression of the Tat transcription factor (a major regulator of transcription encoded by the HIV-1 virus).
  • Serine/threonine phosphatases have recently been studied in Theileria parva, another protozoan close to P. falciparum, a cattle parasite. Monocyte and leukocyte host cells infected by the parasite are transformed, resulting in leukemia in the animal. Purified parasites of cells infected with Theileria express a protein kinase CK2 ⁇ . Now, the subunit CK2 ⁇ should interact with PP2A to positively modulate its activity (Hériché H, et al, Regulation of protein phosphatase 2A by direct interaction with casein kinase 2 ⁇ . Science 1997; 276-952-5).
  • modulation of PP2A via expression of the CK2 ⁇ subunit could be the basis of blockage of two signal routes in the parasitised cell, that of MAP-kinases (Cvicpied M et al. Theileria transformation of bovine leukocytes: a parasite model for the study of lymphoproliferation. Res Immunol 1996; 147: 127-38) and that of protein kinase B (Akt) (M Baumgartner, M Cvicpied, M F Moreau, A Garcia, G Langsley. Constitutive PI3-K activity is essential for proliferation, but not survival, of Theileria parva —transformed B cells. Cellular Microbiol (2000) 2, 329-339).
  • the identification of peptides interacting with PP2A should allow novel drugs to be developed that can block, by competitive inhibition, the cell mechanisms induced by viral or parasitic proteins via their interaction with PP2A and in particular mechanisms of infection, pathogen proliferation and cell transformation.
  • the invention pertains to means for identifying peptides of reduced size, binding a PP2A holoenzyme or one of its subunits.
  • reduced size peptides In contrast to native proteins or polypeptide domains of large size, reduced size peptides have the advantage of being readily synthesized, either chemically or in cell systems, in high yields and cheaply.
  • the peptides of the invention are also more stable and more readily transferred into the cytoplasm or into the nucleus of cells using appropriate vectors, with a view to therapeutic use.
  • the invention derives from the demonstration that it is possible to identify peptides with a size of less than 30 amino acids, and in particular peptides less than 20 amino acids in size, interacting with a PP2A holoenzyme or one of its subunits.
  • the inventors have identified peptides less than 20 amino acids in size interacting in vitro with purified PP2A holoenzyme or one of its subunits, said peptides being derived from the Vpr protein of HIV-1 or the CK2 ⁇ protein of the T parva parasite. Antagonists derived from these peptides and selected because they inhibit the interaction of viral or parasititc proteins with a particular PP2A holoenzyme could then constitute novel anti-tumoural, antiviral or antiparasitic agents.
  • the invention concerns a method for identifying a peptide the sequence of which is derived from a viral, parasitic or cellular protein, said peptide specifically binding a type, 2A protein phosphatase holoenzyme or one of its subunits, said method comprising the steps consisting of:
  • step a different peptides are deposited on a solid support in defined positions (“spot”), each position corresponding to a specific peptide sequence and the series then forming a two-dimensional array of peptides.
  • spot defined positions
  • Different methods for preparing such arrays have recently been described (for a review, see Figeys and Pinto, 2001, Electrophoresis 22: 208-216; Walter et al, 2000, Curr Opin Microbiol 3: 298-302).
  • the series of these methods generally include covalently fixing the peptides on a support, in particular using chemical linkers.
  • the skilled person could refer to the “SPOT synthesis” technique consisting of directly synthesizing peptides comprising up to 20 residues on a cellulose membrane (Frank and Overwing, Methods in Molecular Biology, 1996, vol 66: 149-169, Epitope Mapping Protocols, edited by: G E Morris, Humana Press Inc, Totowa N.J.).
  • any method can be used provided that it can produce an array of peptides deposited on a solid support that can be used to detect specific interactions between the deposited peptides and particular compounds.
  • the series of deposited peptide sequences covers the complete sequence of the viral, parasitic or cellular protein from which those sequences are derived.
  • the process can test, in a single step, the complete sequence of a given protein, this being “sectioned” into a finite number of peptides with generally overlapping sequences.
  • the peptides deposited in the form of a spot are less than 20 amino acids in size, and more preferably are less than 15 amino acids in size.
  • the peptides are deposited on a cellulose membrane.
  • the array obtained is brought into contact in step b) with a type 2A protein phosphatase holoenzyme or one of its subunits.
  • type 2A protein phosphatase holoenzyme means any purified dimeric (AC) or heterotrimeric (ABC) complex of a cellular or reconstituted extract after purifying two subunits (A) and (C) of a type 2A protein phosphatase and if necessary a subunit (B).
  • the type 2A protein phosphatases are preferably derived from mammals.
  • the supports are incubated, for example, in a buffer solution comprising purified protein phosphatase or one of its purified subunits.
  • a suitable buffer solution is TBS (TRIS BORATE) containing 5% of skimmed Regilait (milk) and 3% of BSA.
  • the peptide onto which the type 2A protein phosphatase holoenzyrne is bound is generally identified by direct or indirect labeling of the protein phosphatase and identifying the spots to which the labeled protein has bound. Binding of PP2A or one of its subunits to one of the peptide spots can then be revealed, in particular using antiserums, using techniques that are conventionally used for Western Blot or solid phase ELISA test, after incubating the support containing the peptide array with an antibody directed against subunits (A) or (B) or (C) or a mixture of antibodies directed against subunits (A), (B) or (C) of PP2A.
  • the method of the invention can be applied to identifying peptides, in particular for use in treating certain viral or parasitic infections, measuring less than 30 amino acids in size or even less than 20 amino acids, said peptides being capable of binding a type 2A protein phosphatase holoenzyme or one of its subunits in vitro.
  • the invention provides a natural or synthetic peptide measuring less than 30 amino acids, preferably less than 20 amino acids, characterized in that in vitro, it specifically binds a type 2A protein phosphatase holoenzyme or one of its subunits (A), (B) or (C).
  • the term “specifically binds” means that the peptide is capable of competitively inhibiting binding of a protein of viral or parasitic origin with PP2As.
  • the peptide of the invention is characterized in that it is a fragment of a viral, parasitic or cellular protein, said protein binding in vitro a type 2A protein phosphatase or one of its subunits, or a sequence that is distinguished from the preceding protein fragment by substitution or deletion of amino acids, said distinct sequence nevertheless conserving the properties of binding to the type 2A protein phosphatase or one of its subunits.
  • the number of amino acids substituted or deleted from the distinct sequence compared with the initial sequence does not exceed 20%, more preferably 10% of the amino acids number constituting the initial sequence.
  • only amino acids the deletion of which does not affect the in vitro binding properties of the peptide to PP2A are substituted or deleted.
  • one distinct sequence is a peptide sequence increasing the binding affinity to type 2A protein phosphatase or one of its subunits compared with the sequence from which it is derived.
  • a further distinct sequence as defined above is a peptide sequence homologous with an initially identified peptide sequence.
  • the term “homologous peptide” as used in the present invention means a sequence derived from a protein of species other than the initially identified peptide sequence, and for which the primary sequence can be aligned with the peptide sequence initially identified using a conventional optimum alignment program such as the BESTFIT program (Wisconsin Genetics Software Package, Genetics Computer Group, GCG).
  • a sequence A will be considered to be homologous with a sequence B if said sequences A and B have at least 50% identity, preferably 75% identity after aligning the sequences using an optimum alignment program such as the BESTFIT program.
  • two sequences are also considered to be homologous if the sequences are quasi-identical, with the exception of a few residues that can represent 10% to 20% variability over the whole sequence.
  • amino acids with the same chemical function such as Arg and Lys
  • the peptides to be analyzed for their binding with a PP2A or one of its subunits are generally selected from fragments of viral, parasitic or cellular proteins, which proteins have been shown to interact in vivo or in vitro with a type 2A protein phosphatase.
  • viral parasitic or cellular proteins are selected from one of the following proteins: the t antigen of SV40 or polyoma, the middle t antigen of polyoma, the type B (B, B′, B′′) subunit of PP2A, CK2 ⁇ , CaMIV, p70S6-kinase, Pak1/Pak3, Tap42/alpha 4, PTPA, Set/I1/I2-PP2A, E4orf4, tau, Vpr or CD28, CCXR2 (chemokine receptor).
  • a preferred peptide of the invention is a fragment of the CD28 protein, and in particular peptides constituted by the sequences PRRPGPTRKHY (SEQ ID No: 132) and (PRRPGPTRK) 2 (SEQ ID No: 133), respectively corresponding to the peptides termed FD2 and FD3 the intracellular penetration capacity and effects on cell viability of which are described below in the experimental section.
  • the present invention also pertains to peptide sequences that are distinguished from the preceding protein by substitution or deletion of amino acids, said distinct sequences nevertheless conserving the properties of binding to type 2A protein phosphatase or one of its subunits.
  • a particularly preferred peptide of the invention is a fragment of the Vpr protein of the HIV virus, in particular a fragment of the Vpr protein of the HIV-1 or HIV-2 virus, or a sequence that is distinguished from the preceding protein fragment by substitution or deletion of amino acids, said distinct sequence nevertheless conserving the properties of binding to type 2A protein phosphatase or one of its subunits.
  • the invention does not encompass the peptide, a fragment of the Vpr protein having the following sequence: LFIHFRIGCQHSRIGITRRRRVRDGSSRP* disclosed in the EMBL database, accession number P89821.
  • using said peptide in the context of the applications described below falls within the scope of the present invention.
  • peptides derived from a protein which interacts with type 2A protein phosphatase derived from protamine are the peptide with sequence RRRRRRRSRGRRRRTY (SEQ ID No: 140, termed FD8) or a sequence that is distinguished from SEQ ID No: 140 by substitution or deletion of amino acids, said distinct sequence nevertheless conserving the properties of binding to type 2A protein phosphatase or one of its subunits.
  • a peptide of the invention is characterized in that it is included in one of the following sequences:
  • a particularly preferred peptide of the invention is a fragment of the peptide SEQ ID No: 2, said fragment consisting of or comprising the peptide with sequence RHSRIG (SEQ ID No: 135), termed FD9, the capacity for intracellular penetration and the effect on cell viability of which are described below in the experimental section.
  • the invention also concerns a compound with a polypeptide framework containing a peptide of the invention as defined above, said compound having a molecular weight in the range 10 to 150 Kdaltons and having the capacity to bind protein phosphatase 2A.
  • the invention also concerns a polypeptide, characterized in that it is constituted by a repeat of a peptide of the invention.
  • polypeptides are the peptide RHSRIG polymers, and in particular the dimer (RHSRIG) 2 (SEQ ID No: 136) or the trimer (RHSRIG) 3 (SEQ ID No: 137), respectively termed FD10 and FD11, the capacity for intracellular penetration and the effect on cell viability of which are described below in the experimental section.
  • Peptides with sequences that are distinguished from SEQ ID No: 1 or SEQ ID No: 2 by substitution or deletion of amino acids and falling within the scope of the invention that can in particular be cited peptides the sequence of which is included in one of the sequences for the Vpr protein of different variants of type HIV-1, HIV-2 and SIV, corresponding to homologous sequences in variants of SEQ ID No: 1 or SEQ ID No: 2.
  • VEALIRILQQLL SEQ ID No: 6
  • ALIRILQQLLFI SEQ ID No: 7
  • IRILQQLLFIHF SEQ ID No: 8
  • ILQQLLFIHFR SEQ ID No: 9
  • RHSRIGIIQQRR SEQ ID No: 10
  • SRIGIIQQRRTR SEQ ID No: 11
  • IGIIQQRRTRNG SEQ ID No: 12
  • a particular sequence of the invention that is distinguished from SEQ ID No: 2 by deletion or substitution of amino acids is the sequence RHSRIGVTRQRRARNG (SEQ ID No: 139), also termed FD13 in the experimental section described below.
  • a preferred peptide of the invention is a peptide selected from sequences SEQ ID No: 1 and SEQ ID No: 2 and is characterized in that its administration induces apoptosis of tumour cells.
  • One method for selecting peptides that can induce tumour cell apoptosis can be implemented, for example, using the MTT viability test described in the experimental section.
  • a further preferred implementation of the invention provides a peptide characterized in that it derives from a fragment of the CK2 ⁇ protein.
  • the natural or synthetic peptide is characterized in that it derives from a fragment of the CK2 ⁇ protein of the Theileria parva parasite.
  • a peptide of the invention is characterized in that it is included in one of the following sequences:
  • sequences from site 1 RKIGRGKFSEVFEG (SEQ ID No: 3), in particular the peptide with the sequence RKIGRGKFSEVF and the peptide with sequence IGRGKFSEVFEG or sequences from site 2 (TVTKDKCVIKILKPVKKKKIKRIKILQNL) (SEQ ID No: 4), in particular the following peptides: TVTKDKCVIKIL; (SEQ ID No: 13) TKDKCVIKLLKP; (SEQ ID No: 14) DKCVIKILKPVK; (SEQ ID No: 15) CVIKILKPVKKK; (SEQ ID No: 16) IKILKPVKKKKI; (SEQ ID No: 17) ILKPVKKKKIKR; (SEQ ID No: 18) KPVKKKKIKREI; (SEQ ID No: 19) VKKKKIKREIKI
  • a peptide of the invention comprising a sequence homologous to T parva from site 3 of the CK2 ⁇ protein in P falciparum is the peptide RQKRLI (SEQ ID No: 141).
  • the invention also encompasses polymers of the peptide RQKRLI and in particular the trimer (RQKRLI) 3 (SEQ ID No: 134), termed FD7 in the experimental section.
  • the invention pertains to a peptide derived from the CK2 ⁇ protein of the parasite Theileria parva, characterized in that its administration reduces parasitic development.
  • a further embodiment of the peptides of the invention is characterized in that the peptides are derived from the tau protein.
  • the tau sequence has a motif corresponding to the binding site for the E4orf4 adenovirus protein.
  • the tau protein is regulated by protein phosphatase 2A. Such peptides should thus be useful in treating Alzheimer's disease.
  • the peptides identified by the method of the invention are particularly useful in treating certain tumours and certain viral or parasitic infections.
  • the skilled person can select, using binding competition tests, novel peptides derived from the sequences identified using the method of the invention, said peptides competitively inhibiting binding of the native protein from which it derives with a holoenzyme PP2A or one of its subunits.
  • the invention also concerns a natural or synthetic peptide as defined above, characterized in that it competitively inhibits interaction of the native protein from which it derives with a PP2A holoenzyme or one of its subunits.
  • the peptides of the invention can be coupled to a vector that is capable of transferring said peptide into a eukaryotic cell.
  • a vector that is capable of transferring said peptide into a eukaryotic cell.
  • the peptides of the invention it is possible, as will be discussed below, for the peptides of the invention to themselves have the capacity to penetrate into cells, meaning that the addition of a vector is not required.
  • the invention pertains to means that can synthesise the peptides of the invention.
  • the invention pertains to a polynucleotide characterized in that its sequence consists of the sequence coding for a peptide of the invention.
  • Preferred polynucleotides are polynucleotides the sequence of which is selected from one of the following sequences: SEQ IDs No: 26 (5′GTGGAAGCCTTAATAAGAATTCTGCAACAACTGCTGTTTATTCATTT CAGAATT); No: 27 (5′CGACATAGCAGAATAGGCATTATTCAACAGAGGAGAACAAGAAATGG A); No: 28 (5′AGGAAGATCGGAAGAGGGAAGTTCAGTGAAGTTTTTGAGGGA); No: 29 (5′ACAGTAACGAAGGATAAATGCGTAATAAAAATCCTAAAGCCTGTAAA GAAGAAGAAAATCAAGAGAGATTAAGATTCTACAGAACCTA); or No: 30 (5′AAAATACTAAGGCTAATTGACTGGGGATTAGCTGA
  • the invention also concerns polynucleotides with sequences complementary to one of sequences SEQ ID No: 26-30 and sequences hybridizing under stringent conditions to said polynucleotides.
  • stringent conditions means conditions that allow specific hybridization of two single strand DNA sequences at about 65° C., for example in a solution of 6 ⁇ SSC; 0.5% SDS, 5 ⁇ Denhardt's, solution and 100 ⁇ g of non specific carrier DNA or any other solution with an equivalent ionic strength and after washing at 65° C., for example in a solution of at most 0.2 ⁇ SSC and 0.1% SDS or any other solution with an equivalent ionic strength.
  • the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
  • the stringency conditions have also been defined using protocols described by Sambrook et al, 2001 (Molecular cloning: a laboratory manual, 3 rd edition, Cold Spring Harbor, Laboratory Press, Cold Spring Harbour, N.Y.).
  • the invention pertains to a polynucleotide characterized in that it consists of a multimer of a polynucleotide coding for a peptide of the invention.
  • the invention also pertains to a polypeptide characterized in that it is constituted by a repeat of a peptide of the invention.
  • the invention also pertains to a cell expression vector, characterized in that it comprises a polynucleotide as defined above and regulatory sequences allowing expression of a peptide of the invention in a host cell.
  • the invention also pertains to a method for preparing a peptide as defined in the invention, comprising transforming a host cell using a cellular expression vector as defined above, followed by culturing the transformed host cell, and recovering the peptide in the culture medium.
  • the invention further pertains to an antiserum or immunoserum or a purified polyclonal antibody or a monoclonal antibody, characterized in that said antibody or said antiserum or immunoserum is capable of specifically binding a peptide in accordance with the invention.
  • Antibodies specifically directed against the peptides identified by the process of the invention are obtained, for example, by immunizing an animal after injecting a peptide of the invention, and recovering the antibodies produced.
  • a monoclonal antibody can be obtained using techniques that are known to the skilled person, such as the hybridoma method describd by Kohler and Milstein (1975).
  • the antibodies obtained are of particular application in immunotherapy.
  • they can act as antagonists for viral or parasitic proteins directed against protein phosphatase 2A to block viral or parasitic development.
  • polynucleotides encoding the peptides of the invention can be directly transferred to the nucleus of target cells, if necessary using suitable vectors, to allow in vivo expression of the corresponding peptides, said peptides being susceptible of blocking by competitive inhibition a specific interaction between the protein phosphatase 2A and the viral or parasitic protein from which they derive.
  • the invention thus pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising one of the elements selected from a polynucleotide of the invention or an antibody of the invention.
  • the invention also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising one of the peptides of the invention in combination with a pharmaceutically acceptable vehicle.
  • the invention further concerns the use of a peptide of the invention as defined above in preparing a drug for use in treating a viral or parasitic infection.
  • the invention concerns the use of a peptide the sequence of which derives from a fragment of the Vpr protein as defined above, in preparing a drug that can inhibit an HIV infection.
  • the peptides of the invention can advantageously be selected so as to stimulate the induction of apoptosis linked to activation of cellular protein phosphatase 2A.
  • the invention also concerns the use of a peptide of the invention as defined above in preparing a drug that can induce apoptosis of target cells and in particular tumour cells.
  • the invention concerns the use of a peptide the sequence of which derives from a fragment of the CK2 ⁇ protein in preparing a drug that can inhibit parasitic infection. More particularly, the invention concerns the use of a peptide in preparing a drug for use in treating malaria.
  • the viral or parasitic infection results in specific expression of proteins comprising sequences of peptides of the invention.
  • the sequences encoding the peptides of the invention can thus be used as a probe to detect, in a specific manner from RNA extracted from a biological sample from a patient, a specific viral infection or parasitic infection.
  • an antibody of the invention can be used to specifically recognize peptide sequences contained in viral or parasitic proteins expressed during infection.
  • the invention concerns the use of a polynucleotide of the invention or an antibody of the invention in the in vitro diagnosis of parasitic or viral diseases.
  • the invention also pertains to the selection and use of a peptide binding protein phosphatase 2A, and capable of penetrating into cells.
  • peptide FD6 SEQ ID No: 20
  • peptide FD6 SEQ ID No: 20
  • CK2 ⁇ protein of T parva An example of such a peptide is illustrated by the peptide FD6 (SEQ ID No: 20) derived from the CK2 ⁇ protein of T parva. It has been shown in the present invention that the presence of that peptide in the cell does not affect the viability of cultivated or maintained mammal cells alive.
  • the experimental section below illustrates an application of the method for identifying the peptides of the invention to identifying peptides from the Vpr protein of HIV-1 and of the CK2 ⁇ protein from the Theileria parva parasite.
  • the invention also pertains to the selection and use of a peptide binding protein phosphatase 2A and possibly capable of penetrating into a cell, said peptide enabling targeting of and contact with intracellular protein phosphatase 2A of a molecule capable of regulating the activity of protein phosphatase 2A.
  • FIG. 1 Screening of a membrane containing peptides covering the sequence for Vpr of HIV-1 with the structural subunit A of PP2A (A) and the holoenzyme PP2A1 (B).
  • VEALIRILQQLLFIHFRI SEQ ID No: 1
  • Peptide 54 VEALLRILQQLL Peptide 55: ALIRILQQLLFI Peptide 56: IRILQQLLFIHF Peptide 57: ILQQLLFIHFRI
  • RHSRIGIIQQRRTRNG SEQ ID No: 2
  • Peptide 64 RHSRIGIIQQRR Peptide 65: SRIGIIQQRRTR Peptide 66: IGIIQQRRTRNG
  • FIG. 2 Screening of a membrane containing peptides covering the sequence for CK2 ⁇ of Theileria with (A) the structural subunit A of PP2A and (B) the holoenzyme PP2A1.
  • RKIGRGKFSEVFEG SEQ ID No: 3
  • Peptide 66 RKIGRGKFSEVF
  • Peptide 67 IGRGKFSEVFEG
  • Peptide 74 TVTKDKCVIKIL Peptide 75: TKDKCVIKILKP Peptide 76: DKCVIKILKPVK Peptide 77: CVIKILKPVKKK Peptide 78: IKILKPVKKKKI Peptide 79: ILKPVKKKKIKR Peptide 80: KPVKKKKIKREI Peptide 81: VKKKKIKREIKI Peptide 82: KKKIKREIKILQ Peptide 83: KIKREIKILQNL
  • FIG. 3 is a histogram representing the intracellular penetration values obtained using a cell penetration test for the peptides cited in Table 3.
  • FIG. 4 illustrates the effects of different peptides on the viability of Hela cells evaluated using a MTT viability test.
  • Trimeric PP2A1 protein was purified to homogeneity from pig brain.
  • a recombinant structural subunit of PP2A was expressed in E coli and purified using the protocol described by Cohen et al (Cohen P, Alemany S, Hemmings B A, Resink T J, Stralfors P, Tung H Y. Protein phosphatase-1 and protein phosphatase 2A from rabbit skeletal muscle. Methods Enzymol 1988 159, 390-408), or that described by Bosch et al (Bosch M, Cayla X, Van Hoof C, Hemmings B A, Ozon R, Merlevede W, Goris J. the PR55 and PR65 subunits of protein phosphatase 2A from Xenopus laevis. Molecular cloning and developmental regulation of expression. Eur J Biochem 1995, 230, 1037-45).
  • Binding peptides derived from CK2 ⁇ proteins (encoded by T parva protozoa) or Vpr protein (encoded by the HIV-1 virus) with PP2A were identified using the “spot peptides” technique described above (Frank and Overwing, 1996, Meth Mol Biol 66, 149-169).
  • the method consisted of synthesizing dodecapeptides, in situ on a cellulose membrane, at defined positions wherein the series of the sequence covered the whole sequence of the protein of interest (Vpr or CK2 ⁇ ).
  • the peptides of two consecutive spots on the membrane overlap with an overlapped by two amino acids.
  • each prepared membrane was first saturated for 1 hour at ambient temperature with TBS containing 5% skimmed Régilait (milk) and 3% BSA then incubated overnight in the same buffer in the presence of 4 ⁇ g/ml of purified protein (subunit A of PP2A or holoenzyme PP2A1).
  • purified protein subunit A of PP2A or holoenzyme PP2A1
  • the specific interaction of each purified protein (respectively the structural subunit A or the trimeric holoenzyme PP2A1) with a peptide sequence was revealed, as in Western blot, after incubating the membrane with an antibody directed against the structural protein A ( FIGS. 1A and 2A ) and with a mixture of antibodies recognizing the proteins A, B and C of PP2A ( FIGS. 1B and 2B ).
  • the membranes were washed 5 times for 15 minutes with a conventional TBST buffer (TBS+TWEEN) used for incubation then incubated a further 1 hour at ambient temperature with a second antibody (coupled with peroxidase). Finally, the membranes were washed 5 times for 15 minutes with the TBST buffer and revealed.
  • a conventional TBST buffer TBS+TWEEN
  • a second antibody coupled with peroxidase
  • Hela line which is derived from a human cervical carcinoma.
  • Hela cells (10 4 in 100 1) were seeded into 96-well plates (flat bases) with complete DMEM medium in the presence of 2.5% penicillin/ampicillin and 10% foetal calf serum. After incubating overnight at 37° C., in a CO 2 oven (5%), different dilutions of complexes (biotinylated-avidine peroxidase peptides) were added. After incubating for 4 hours the supernatant was aspirated and the cells were washed 3 times with PBS, trypsinated and taken up for counting in PBS. After counting, the cells were taken up in 300 ⁇ l of lysis buffer.
  • Hela cells (10 4 for 100 ⁇ l) were seeded into 96-well plates (flat bases) with complete DMEM medium containing 2.5% penicillin/ampicillin and 10% foetal calf serum. After incubating overnight at 37° C. in a CO 2 oven (5%), the cells were cultivated in the presence of different peptide concentrations. After incubating for 72 h, the medium containing the peptides was aspirated and the MTT at 0.5 mg/ml (diluted in DMEM alone) was added in an amount of 100 per well. Incubation was carried out in the dark at 37° C. for 30 minutes then the MTT was aspirated off and 50 ⁇ l of DMSO was added to all wells.
  • FIG. 1B Two peptide sequences containing a binding site for the Vpr of HIV-1 with the protein PP2A1 ( FIG. 1B ; “site 1”) and with the subunit A ( FIG. 1A , “site 1” and “site 2”) were identified.
  • Three peptide sequences containing a binding site for CK2 ⁇ of T parva with the protein PP2A1 ( FIG. 2B , “site 3”) and with the structural subunit A were also identified ( FIG. 2A , “site 1”, “site 2” and “site 3”).
  • Vpr The exogenic expression or expression due to proviral infection of the Vpr of HIV-1 induces apoptosis in Hela cells, T lymphoid lines and primary lymphocytes (Stewart et al, 1997 J Virol 71: 5579-9).
  • Vpr mutants known in the art indicates that the peptides identified by the process of the invention and specifically binding the PP2A protein contain sequences which correlate with those required for the pro-apoptotic effect of Vpr.
  • fragments of viral proteins, Vpr and E4orf4, which interact with PP2A and are identified by the process of the invention, could be useful in inducing apoptosis of tumor cells.
  • the identified peptides are also naturally used in inhibiting infection by HIV or other related viruses and retroviruses.
  • PP2A controls cell proliferation via a novel cascade of phosphorylations involving PI3-kinase, PKC ⁇ (identified as a MAP-kinase-kinase-kinase or MEKK), the MEK protein and the two MAP kinases ERK-1 and ERk-2, and transcription factors NF- ⁇ B and Sp1 (Sontag E, Sontag J M, Garcia A (1997), EMBO J, 16, 5662-5671; Ayllon V, Martinez A, C, Garcia A, Cayla X and Rebollo A (2000), EMBO J 19, 1-10, A Garcia, S Cereghini, E Sontag (2000), J Biol Chem 275, 9385-9389).
  • FD12 a 18AA peptide the sequence of which derives from that of the FD11 peptide (R is mutated into A).
  • This peptide which is homologous with the glucosamine transferase protein of Chlamydia muridarum, affects the viability of the Hela cell ( FIG. 4C ). This biological effect could be due to an interaction with the plasma membrane.
  • Vpr a protein coded by the HIV-1 virus, is involved in maintaining a high viral charge and in establishing pathogenesis linked to HIV.
  • the expression of Vpr exogenic or due to proviral HIV-1 infection, induces apoptosis in Hela cells, in T lymphoid lines, in primary lymphocytes and in transformed cells (Stewart et al, J Virol 1997, 71, 5579-9; Stewart et al 1999, PNAS, 96, 12039-12043).
  • adenovirus E4orf4 Marcellus et al, J Virol 2000, 74, 7869-7877
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AU2003220222A1 (en) * 2002-03-13 2003-09-29 Signum Biosciences, Inc. Modulation of protein methylation and phosphoprotein phosphate
FR2850396B1 (fr) * 2003-01-29 2005-05-13 Pasteur Institut Vecteurs destines au transfert de molecules d'interet dans des cellules cibles
WO2004011595A2 (fr) * 2002-07-26 2004-02-05 Institut Pasteur Vecteurs destines au transfert de molecules d'interet dans des cellules cibles
WO2005103654A2 (fr) * 2004-04-09 2005-11-03 Bioalliance Pharma Methode d’identification de composes actifs sur la replication du virus hiv.
WO2006084033A1 (fr) 2005-02-03 2006-08-10 Signum Biosciences, Inc. Compositions et procedes pour l'amelioration de la fonction cognitive
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EP2301956A1 (fr) 2009-09-25 2011-03-30 Institut Pasteur Procédés pour le pronostic de la progression ou la non-progression sur le long terme d'une infection par VIH ou de troubles associés au VIH chez un patient
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